Plant health and growth is affected by the chlorophyll pigments and their relationship to photosynthesis in plants. Plants are green because the chlorophyll pigments reflect visible green light and absorb blue and red light from the visible light spectrum. The photon absorbed energy is passed on to two photosynthetic reaction centers, Photosystem II (PSII) and Photosystem I (PSI). The photon absorbed energy in PSII has three competing uses; (1) the increase of photon energy extracts hydrogen electrons from water releasing oxygen to the atmosphere. The electron transport is used to increase photochemistry in the form of photosynthetic chemicals ATP and NADPH which react with absorbed carbon dioxide to produce carbohydrate for the growth and yield of plants; (2) some of the absorbed energy is also emitted in the form of heat and is not part of the photosynthetic process and (3) a low amount of absorbed photon energy is emitted in the form of chlorophyll fluorescence in the red spectral region. These competing uses result in the following. If photochemistry and/or non-photosynthetic heat are active for absorbed energy, fluorescence will be low. If they are inactive, then fluorescence will be high. This simplified description of photosynthesis and the inverse relationship of fluorescence to photosynthetic efficiency has had considerable detailed study in the past. Researchers have sought to detect plant stress responses by measuring the emitted fluorescence and the photosynthesis relationship with the use of various instruments for applications in biology, agriculture and ecology.
The important universal parameter that relates photosynthetic efficiency and health in plants is the yield and lifetime of chlorophyll pigments which absorb light energy in certain visible spectral bands and, as chlorophyll fluorescence, emit part of this energy at longer wavelengths in the far-red and near-infrared spectral regions. Chlorophyll fluorescence will increase if the chlorophyll-absorbed energy exceeds the plant's immediate photosynthetic activity or efficiency. In general, the magnitude of fluorescence emission is inversely proportional to the photosynthetic efficiency of the plant. The fluorescence emission is also an indicator that is directly proportional to the concentration of chlorophyll (yield) in the plant leaf.
Monitoring plant fluorescence emissions provides the opportunity to monitor plant health. A low emission is normal in healthy plants. Abnormal increased fluorescence emissions are caused by external plant stress stimuli that decrease photosynthetic activity or efficiency or damage photosynthesis pathways. If the stress condition is removed, and no photosynthetic damage has resulted, the plant will acclimate to an Early Stress stage and even recover from an Advance Stress stage. If the stress condition continues unabated, the deterioration of photosynthesis will continue and extend to a Critical Stress stage, with the first visible indication (color change or wilt) of photosynthetic pathway damage and a non-recoverable plant stress condition. This disrupts the photosynthesis process where the yield and quanta of chlorophyll molecules decreases, exacerbating the condition to the Lethal stage with extreme visible symptoms) and causes plant death.
Plant stress pressures may be caused by pathogens including worms or viral, bacterial and fungal disease, or from environmental causes including temperature, water drought, chemicals or industrial effluents. Other stressors may be due to plant metabolism and physiological changes due to under-fertilization or over-fertilization. The fate of chlorophyll, i.e. its yield and lifetime, must be taken into account when monitoring plant health and the physiological response to plant stress conditions.
A low-cost measure of early plant stress remains the key objective of laboratory research or ground-based systems for commercial grower application. In most cases, a measurement of chlorophyll is the basis on which a determination of stress is made. Simple metering devices may use solar reflectance or active light to measure chlorophyll fluorescence at a sensitive spectral wavelength and compare it to the fluorescence at a second wavelength outside the region of sensitivity. However, these metering methods do not account for fluorescence changes due to the natural variable distribution of chlorophyll fluorescence (located interveinal) or that caused by the plant stress condition. The plant stress condition will vary, so that the fluorescence emission is not uniformly distributed on plants or plant leaves. Fluorescence usually begins at the outer rim of a leaf and on the upper leaves where more photosynthetic activity is located. A limitation of present metering instruments is the inability to spatially locate the varied distributed emission for accurate measurement.
Airborne and satellite remote sensing of vegetation first used passive solar reflectance to measure chlorophyll changes. When plants are measured with a suitable radiometer, the blue reflectance (450–480 nm) and red reflectance (620–700 nm) will be slight, the green reflectance (500–550 nm) will increase and the near infrared reflectance will be greater. The changes are due to the absorption of light by the chlorophyll pigments. Any physiological stress, disease, nutrient or reduced amount of photosynthetic pigments causes an increase in the blue and red reflectance and a substantial decrease in the near infrared reflectance. Data obtained from various spectral ranges and developed as ratios such as NIR/R and NIR−R/NIR+R have been used as vegetation indices to assess plants from airborne or satellite remote sensing platforms. Changes in these ratios can be a relative estimate of stress when data from different areas of the field are compared, even if the specific cause for the stress cannot be identified.
An important limitation of solar passive reflectance is the variation in solar radiation in one location due to atmospheric conditions and/or sun angle and/or that caused by plant orientation. The plant's diurnal changes due to transpiration in the morning are a cause of additional water stress. High humidity, and the presence of dew on leaves, also influence the spectral reflectance. The spectral signature is also influenced by the amount of pigments, leaf angle, leaf texture, the physiological factors of stress, and the plant growth stage. These limitations account for the number of different vegetation indices and relate to the difficulty in correlating a spectral ratio number to the broad range of plant vegetation response.
The remote sensing of vegetation exhibits sharp reflectance changes in the 690–740 nm range. This phenomenon has encouraged the use of narrow-band, multi-spectral radiometry to isolate the two signatures for plant stress response. The analysis of red reflectance, (690 nm), and the ratio of red to NIR reflectance, (690/740), has been shown to be responsive to the status of chlorophyll in the vegetation canopy and in individual plants.
To improve the chlorophyll measurements and remove the limitations of reflectance measurements, ground-based systems have sought the use of active light sources. Active chlorophyll fluorescence measurements use actinic (photosynthetic active) light sources to induce the kinetics of electron transport and to measure chlorophyll fluorescence. Filtered light sources of low wavelength pass-band in the near UV or visible range discriminate the low-level, fluorescence signals from out-of-band noise, and with Fabry-Perot interference filters, detect and improve the resolution for specific chlorophyll fluorescence signatures, usually in the NIR. Laser Induced Fluorescence (LIF) methods use laser light as the light source in a number of science studies. McMurtrey and Chappelle describe LIF using laser light in the NUV (355 nm) to irradiate plants and detect fluorescence signatures at blue, 440 nm; green, 520 nm; red, 690 nm and far-red, 740 nm. The ratios of 690/740, 690/520 and 690/440 are used to determine plant stress in the field (in-situ). Laser Induced Fluorescence Imaging (LIFI) studies by DOE use a laser light source with a line-scan imager to detect chlorophyll fluorescence imaging for sensing with airborne helicopter at night for the detection and measure of induced plant stress (arsenic). Lichtenthaler et al use LIFI to measure the same ratios of non-chlorophyll and chlorophyll fluorescence values at 440/690 or 440/740 to determine plant stress. LIFI methods, with high frequency, high energy NUV lasers, remain a deterrent for commercial application to consumers and non-scientific users.
The measure of low-level chlorophyll fluorescence in the field is difficult in daylight unless the plants or leaf parts are shrouded from ambient light scatter. To overcome this limitation, researchers have sought means to provide a better measure of fluorescence in the field. McFarlane et al described a Fraunhofer line discriminator using 656 nm to measure chlorophyll fluorescence in wet vs. dry water stress in trees. Fraunhofer lines are observed as dark spectral lines of solar light indicating their opacity due to the absorbance by gases in the sun's or earth's atmosphere. The line at 656 nm is due to hydrogen absorption in the solar atmosphere. The Fraunhofer A, B lines at 686 nm and 759 nm are due to the absorption by oxygen in the earth's atmosphere and overlap the spectral bandwidth for the emission of chlorophyll fluorescence. When recording measurements from the vegetative canopy with a spectral detector such as a photo-multiplier, the difference signal at the Fraunhofer center wavelength compared to the adjoining spectral band is chlorophyll fluorescence without any additional out-of-band noise-signal from light scatter. However, fluorescence can reflect and light scatter onto an adjacent plant leaf. To assure that the fluorescence measurements of a single plant leaf are correct, a caveat requires that the plant being measured must be isolated and not affected by the fluorescence/reflectance emission of adjacent plants or leaves.
Kebabian in U.S. Pat. No. 5,567,947 cites the use of Fraunhofer wavelengths at 690 nm and 760 nm to measure chlorophyll fluorescence emissions from vegetative canopy. The Fraunhofer lines, due to the absorption by oxygen in the earth's atmosphere, will eliminate noise from out-of-band light scatter in these bands, leaving only chlorophyll fluorescence and the in-band emissions from the canopy. The method focuses light from the vegetation canopy with a lens and narrow-band filters as input to a quartz tube of oxygen in a spherical cavity, and measures a delayed, secondary emission of oxygen fluorescence at 760 nm that is proportional to the fluorescence/reflected light levels from the vegetative canopy.
In the above references, a simple ratio of reflectance or chlorophyll fluorescence tested in two spectral bands is used to detect the plant stress response. However, single number ratios provide no additional information related to the temporal-related values of fluorescence emission to diagnose the plant stress damage stage.
Lussier in U.S. Pat. No. 5,130,545, describes a Video Plant Management System, using active Chlorophyll Fluorescence Imaging from absorbed light, 400 to 600 nm, with NIR video to image and record the time-dependent chlorophyll fluorescence signatures to detect plant stress. The system uses dark-adapted plants and shutters light to induce transient fluorescence emission (the Kautsky Effect and chlorophyll fluorescence quenching) that is directly attributed to photosynthetic electron transport and energy transfer in the plant's chloroplast cells. The method uses video data (frames per second) to record visually and temporally the time dependent fluorescence emitted to determine the plant's physiological response to disease and plant stress. The method uses a delayed timing to image and measure the chlorophyll fluorescence intensity across a leaf with a line scan, and records the temporal changes of the line scan according to the video rate.